Other

Part:BBa_J100028

Designed by: Malcolm Campbell   Group: Campbell_M_Lab   (2011-08-30)

placeholder insert for BsaI Golden Gate Assembly of promoter

This insert can be placed in any vector that does not contain a BsaI site. BsaI is a type IIs restriction enzyme use in Golden Gate Assembly of multiple inserts. Most versions of ampicillin resistance in plasmids contain a BsaI site. We are putting this insert into a modified pSB1A2 that has its vector BsaI site mutated (see Part:pSB1A2). A similar part (Part:BBa_J119022) has been cloned into pSB1A8 (Part:BBa_J119043) and should be used in place of J100028. This insert can be digested with BsaI and mixed with ligase to make room for a promoter of your choice that has appropriate sticky ends. We are using this insert in combination with new promoters assembled from oligonucleotides (oligos). When assembled, the new promoter would have a four base overhang of 5' GCTG 3' on the left side of the top strand and a four base overhang of 3' CGCC 5' on the right side of the bottom strand:
5' CGACNNN...NNN_____ 3'
3' _____NNN...NNNCGCC 5'

Below is a picture of what this part will look like when digested with Bsa I. TT represents the transcriptional terminator Part:BBa_B0014 J100028.png


This insert contains BBa biobrick prefix, BbsI site, 4 base overhang followed by BsaI site cutting to the left, transcriptional terminator Part:BBa_B0014, BsaI site that cuts to the right, 4 base overhang, BbsI site, RBS Part:BBa_B0034, mRFP Part:BBa_E1010 and ending in BBa biobrick suffix.

Sequence and Features

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 857
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal SpeI site found at 858
    Illegal PstI site found at 872
    Illegal NotI site found at 7
    Illegal NotI site found at 865
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 858
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 858
    Illegal PstI site found at 872
    Illegal AgeI site found at 730
    Illegal AgeI site found at 842
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 135
    Illegal BsaI.rc site found at 34
    Illegal EcoRI site found at 1
    Illegal XbaI site found at 16
    Illegal SpeI site found at 858
    Illegal PstI site found at 872


[edit]
Categories
Parameters
None